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F U L L T E X T S O U R C E : Development
However, more recent studies have uncovered beneficial effects of senescence, for example in the context of embryonic development, tissue repair/regeneration, and cellular reprogramming. As we review here, these discoveries have helped to broaden our understanding of the biological functions of the senescence process.
The senescence program
A primary feature of senescence, which separates it from quiescence or cell-cycle arrest, is a state of irreversible proliferative withdrawal. In tissue culture, senescent cells often exhibit a large flattened morphology, sometimes having multiple nuclei and large vacuoles. However, these size and shape changes may not occur in the same way in tissues. An additional feature of senescent cells is that they are resistant to apoptosis-inducing stimuli, a factor that likely contributes to their survival (Baar et al., 2017; Chang et al., 2016; Yosef et al., 2016). At the molecular level, the senescence program consists of two main components – the intrinsic arm and the extrinsic arm – that are broadly activated irrespective of the inductive stimulus, but exhibit some context-specific features, as discussed below (Fig. 1).
Fig. 1
Overview of the cellular senescence program. Senescence can be induced in response to a variety of inducers (left). Once activated, the senescence program then involves a number of key factors (‘mediators’) that mediate both the intrinsic and extrinsic arms of the senescent response. The intrinsic arm includes the tumor suppressor genes p53, p21, p16INK4A and p19ARF. These help to establish the cell cycle arrest and coordinate the complex senescence program. Furthermore, the activation of transcription and signaling factors including Nfkb, Cebpß, Gata4 and p38, in addition to p53, controls the extrinsic arm, a key part of which is known as the ‘senescence-associated secretory phenotype’ – the secretion of a cocktail of proteins by senescent cells that enables their interaction with the neighboring environment.
The intrinsic arm
The intrinsic arm regulates cell cycle arrest and is broadly mediated by key regulatory proteins including the p53 (also known as Trp53 or TP53), p21 (Cdkn1a), p16INK4A and p19ARF (both encoded by the Cdkn2a locus) tumor suppressors, which act to block the cell cycle and establish the irreversible arrested state (Kuilman et al., 2010; Martínez-Zamudio et al., 2017; Narita et al., 2003; Serrano et al., 1997). Senescence arrest is also fine-tuned by microRNA-mediated gene silencing (Benhamed et al., 2012). Some inducers of senescence also cause DNA damage, so immunostaining for markers of DNA damage such as γH2AX and 53BP1 can be used in some cases to identify senescent cells. However, it should be noted that no single marker can be used to identify all senescent cells (see Box 1).
Box. 1. Markers of senescence
One of the main challenges in senescence research is that there is currently no single marker that can be used to identify all senescent cells. The most commonly used is ‘senescence-associated β-galactosidase’ (SA-ß-gal), which makes use of the increased amount and activity of the enzyme β-galactosidase in enlarged lysosomes, which catalyzes a color reaction in cells at lower pH, turning them blue in the presence of X-gal (Dimri et al., 1995). This is analogous to the staining of lacZ reporter mice, but at a lower pH (5.5) and in the absence of a transgene, using instead the endogenous β-galactosidase gene Glb1. However, it is possible to have senescent cells that do not stain with SA-ß-gal, as demonstrated in cells lacking Glb1 (Lee et al., 2006) and, for example, in mouse papilloma (Ritschka et al., 2017). Conversely, it is possible to have false-positive staining from macrophages (Hall et al., 2017). In addition, it appears that some tissues in the embryo stain positive for SA-ß-gal while not expressing other key markers such as p21 (Huang and Rivera-Pérez, 2014). Therefore, caution and diligence are needed when claiming senescence identification and, at a minimum, cells that are suggested to be senescent should exhibit a combination of senescence markers and features. Importantly, efforts are ongoing to identify potential new markers of senescence, including cell-surface proteins (Althubiti et al., 2014; Kim et al., 2017; Sagiv et al., 2016), commonly expressed senescence genes (Hernandez-Segura et al., 2017; Wiley et al., 2017) and histological stains (Evangelou et al., 2017).
Complex changes in 3D chromatin organization within the nucleus, as well as epigenetic changes, also occur in senescent cells. Changes in the nuclear lamina, including loss of lamin B1, occur in many states of senescence, and are suggested to enable spatial rearrangement of heterochromatin (Freund et al., 2012). In some cases of senescence, the formation of heterochromatin complexes known as senescence-associated heterochromatin foci (SAHF) is observed. These complexes consist of repressive chromatin regulators and marks, including HP1, MacroH2A, H3K9me3 and H3K27me3, concentrically layered to repress proliferation-associated genes and condense chromosomes. These epigenetic- and chromatin-mediated changes occurring in and regulating senescence have recently been reviewed in detail (Parry and Narita, 2016).
The extrinsic arm of the senescence program consists of the ‘senescence-associated secretory phenotype’ (SASP). This is a hallmark feature of senescent cells that reflects their ability, even though they are arrested from proliferation, to produce a rich secretome to interact with their external environment (Coppé et al., 2008). Although a detailed understanding of the composition of the SASP is still emerging, it is broadly composed of growth factors, cytokines, chemokines, and extracellular matrix (ECM) and ECM-remodeling proteins (Acosta et al., 2013; Coppé et al., 2010b; Freund et al., 2010) (Fig. 1). The regulation of this secretion is also highly coordinated and dynamic. Primary transcriptional regulators of the SASP include the Nfkb, Cebpβ, p53 and Gata4 transcription factors (Acosta et al., 2008; Kang et al., 2015; Kuilman et al., 2008), but also p38 MAPK, which regulates a DNA damage-independent SASP (Freund et al., 2011), and Notch1, which orchestrates a switch in SASP composition during senescence onset (Hoare et al., 2016). In addition, there is a pronounced epigenetic regulatory component to SASP control, with MLL1 (KMT2A), HMGB2, H2A.J and MacroH2A relocalization occurring early after senescence induction to regulate SASP gene expression (Aird et al., 2016; Capell et al., 2016; Chen et al., 2015; Contrepois et al., 2017). Although it is not yet known how SASP composition differs precisely in response to different stimuli, or between cell types, it is clear that the strength and mode of senescence induction is reflected in the SASP. For example, senescence induced by oncogenes such as the Ras genes, or following DNA damage, results in a more pronounced SASP than that induced by other factors (Coppé et al., 2008; Rodier et al., 2009).
Why senescent cells secrete such a rich cocktail of factors has been the subject of many studies. Initially, primary functions attributed to the SASP included reinforcement of cell cycle arrest by cytokines such as IL6 or IL8 via the CCR2 receptor (Acosta et al., 2008; Kuilman et al., 2008). Further, it was found that chronic exposure to the SASP can induce senescence in a paracrine manner in neighboring cells (Acosta et al., 2013). Functionally, some SASP proteins such as Csf1, Ccl2 and IL8 (Cxcl15) promote the recruitment of immune cells, including macrophages and natural killer (NK) cells, which remove senescent cells (Krizhanovsky et al., 2008; Lujambio et al., 2013; Xue et al., 2007). Such functions are in agreement with reinforcing the tumor suppressive role of senescence. Recently, additional cellular features of senescent cells and SASP regulation have been described, including the budding-off of chromatin fragments from senescent nuclei (Ivanov et al., 2013). Interestingly, these senescence-associated nuclear fragments are recognized by the anti-viral defense response, activating the cGAS-STING pathway, which contributes to SASP control (Dou et al., 2017; Gluck et al., 2017; Yang et al., 2017). However, additional effects of the SASP have also been discovered, such as the ability to induce proliferation,angiogenesis or epithelial-mesenchymal transition (EMT) in neighboring or cancer cells (Coppet al., 2010a, 2006; Gonzalez-Meljem et al., 2018; Krtolica et al., 2001). Together, these effects have suggested broader biological roles for senescent cells and the SASP, which are harder to reconcile with a simple tumor suppressive or aging function.
Senescence in disease and aging
Much of what we know about the role of senescence comes from in vivo genetic manipulation of either the cell-intrinsic or the cell-extrinsic aspects of senescence in different contexts of cancer and aging. In 2005, a series of studies demonstrated that oncogenic mutations in different contexts activate senescence in vivo, as had been previously shown in cells in culture, and that pre-malignant lesions, including papilloma or adenomas in the skin, lung, pancreas, lymphoma and prostate, form through an accumulation of senescent cells (Braig et al., 2005; Chen et al., 2005; Collado et al., 2005; Lazzerini Denchi et al., 2005; Michaloglou et al., 2005). Furthermore, these studies demonstrated that inactivation of the cell-intrinsic senescence machinery, through loss of function of key senescence genes such as p53, p16INK4A or p19ARF, prevents full senescence arrest and allows senescence bypass and malignant progression. These findings supported the notion that senescence is a tumor-suppressive barrier to cancer formation, and demonstrated how the most frequently mutated tumor suppressors in human cancers help protect from cancer by inducing senescence. In support of this, subsequent elegant studies have reported that the re-expression of p53 within p53-deficient solid tumors leads to reactivation of senescence in tumor cells (Xue et al., 2007). Interestingly here, the induced senescent tumor cells also activate an SASP and are actively removed by the immune system, showing how both the cell-intrinsic and cell-extrinsic arms of senescence can have tumor suppressive function (Lujambio et al., 2013; Xue et al., 2007).
However, although the regulated induction of senescence is beneficial in preventing tumor formation, prolonged aberrant persistence of senescent cells can have detrimental effects in promoting cancer. For example, if the timely clearance of OIS cells by the immune system is perturbed, this leads directly to tumor formation (Kang et al., 2011). Similarly, although chemotherapy can, in part, exert beneficial effects by inducing tumor-cell senescence (Schmitt et al., 2002), the persistence of therapy-induced senescent cells can, via the SASP, promote tumor recurrence and metastasis (Demaria et al., 2017; Zacarias-Fluck et al., 2015).
The senescence process has long been linked to aging, including in the original study demonstrating that aging human skin has increased numbers of cells that are positive for the senescence marker senescence-associated beta-galactosidase (SA-ß-gal) (Dimri et al., 1995). In addition, the de-repression of senescence mediators including p16INK4A occurs during chronological aging, and contributes to loss of regenerative capacity in many tissues (Bracken et al., 2007; Krishnamurthy et al., 2006; Krishnamurthy et al., 2004; Sousa-Victor et al., 2014). In recent years, perhaps the most conclusive data linking senescence with organismal aging has come from the use of senescence ‘deletor’ mouse models, in which cells expressing p16INK4A are selectively targeted for elimination (Baker et al., 2016, 2011). In such models, the removal of senescent cells results in significant improvements in health and vigor, and also in lifespan. These studies unequivocally demonstrate how the accumulation of senescent cells during aging can have a negative impact on health and lifespan. Although these effects were primarily shown in response to targeting the cell-intrinsic program, it is likely that the SASP is also diminished in these models.
Similar studies using senescence-ablation mouse models have uncovered detrimental effects of senescent-cell accumulation in many other diseases, including osteoarthritis (Jeon et al., 2017), osteoporosis (Farr et al., 2017), atherosclerosis (Childs et al., 2016), Parkinson’s (Chinta et al., 2018), Alzheimer's (Bussian et al., 2018; Musi et al., 2018) and others, whereas the selective deletion of p16INK4A-positive cells improves many disease symptoms. The use of such models has spurred the search for new strategies to eliminate senescent cells, including drugs (‘senolytics’) and nanoparticles that eliminate senescent cells (‘senotherapy’), which have been found to improve health or aging in many cases (Baar et al., 2017; Chang et al., 2016; Muñoz-Espín et al., 2018; Schafer et al., 2017; Xu et al., 2018; Yosef et al., 2016). In addition, new and previously known drugs are being investigated as SASP modulators for potential therapeutic uses, including glucocorticoids, metformin, Jak/Stat inhibitors and others (Soto-Gamez and Demaria, 2017), and possibly even small molecules targeting STING (also known as Tmem173) (Haag et al., 2018). Together, such approaches highlight how the accumulation of senescent cells can be detrimental to health and demonstrate the beneficial effects of senescent cell elimination or manipulation.
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F O R T H E R E S T O F T H E A R T I C L E , P L E A S E V I S I T T H E S O U R C E .
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